1. Field of the Invention
This invention relates to a microscope equipped with a reflecting fluorescence illumination optical system, and in particular, to a stereomicroscope and a reflecting fluorescence microscope which are designed to prevent a reduction in contrast of an observation image, caused by auto-fluorescent light emitted from a stage mounting a specimen and the optical system of a transmitting illumination device in a fluorescence observation.
2. Description of Related Art
A stereomicroscope, in contrast with an ordinary optical microscope, is very long in working distance and has the feature of being usable for a stereoscopic observation. This feature is utilized for observing a specimen.
As shown in FIG. 1, a conventional stereomicroscope, as its observation optical system, includes an objective lens 1, zoom lenses 2R and 2L, imaging lenses 3R and 3L, and eyepieces 4R and 4L. A specimen 7 is magnified by the objective lens 1 and the zoom lenses 2R and 2L, and images Q and Q' of the specimen 7 are formed by the imaging lenses 3R and 3L so that the images Q and Q' are observed through the eyepieces 4R and 4L.
Of the observation optical system, the zoom lenses 2R and 2L, the imaging lenses 3R and 3L, and the eyepieces 4R and 4L are arranged in pairs for the right and left eyes. An observation optical axis 5R for the zoom lens 2R, the imaging lens 3R, and the eyepiece 4R which constitutes an observation optical path R for the right eye, as well as an observation optical axis 5L for the zoom lens 2L, the imaging lens 3L, and the eyepiece 4L which constitutes an observation optical path L for the left eye, is decentered with respect to an optical axis 6 of the objective lens 1.
For the observation of the specimen, such a stereomicroscope has been used to observe the transmitted image or reflected image of the specimen in most cases. Hence, a transparent member, such as glass, has been used for a stage mounting the specimen 7 when the transmitted image of the specimen is observed. This is because it is necessary to make an arrangement so that the specimen interposed between the object lens and the stage can be illuminated from the opposite side of the object lens with respect to the specimen. Where the reflected image of the specimen is observed, on the other hand, it is only necessary to illuminate the specimen from the objective side, and thus an opaque member has been used as the stage.
Illumination devices vary with observation techniques. A transmitting illumination device used when the transmitted image is observed varies in technique from a simple structure to a complicated illumination system with a means for visualizing a transparent specimen. The stereomicroscope shown in FIG. 1 is a stereomicroscope equipped with a transmitting illumination device disclosed in Japanese Utility Model Preliminary Publication No. Hei 3-39712. In FIG. 1, the optical system of a transmitting illumination device A is composed of a light source 9, a collector lens 10, a Fresnel lens 11, and a deflection mirror 12. Illumination light emitted from the light source 9 which is a halogen lamp is collected by the collector lens 10 and is incident on the Fresnel lens 11. The illumination light is changed to a light beam such that the specimen is almost uniformly illuminated by the Fresnel lens 11, and after being reflected by the deflection mirror 12, reaches the specimen 7 through a glass stage 8. Here, if the light beam illuminating the specimen is spread, the specimen can be obliquely illuminated by rotating the deflection mirror 12, so that a transparent specimen can be visualized by so-called oblique illumination. In the transmitting illumination device of such a conventional stereomicroscope, it is customary to use inexpensive resin material for the Fresnel lens 11 in order to keep cost to a minimum.
Where the reflected image of the specimen is observed, on the other hand, a reflecting illumination device in which an annular illumination light source is placed around the objective lens, or a reflecting illumination device such as that shown in FIG. 2 is used. A reflecting illumination device B shown in FIG. 2 uses a light guide fiber, and its optical system is constructed with a light source 14, a collector lens unit 15, a light guide fiber 16, and an illumination lens unit 17 whose illumination range is variable.
Illumination light emitted from the light source 14 is rendered incident on an entrance end face 16a of the light guide fiber 16 by the collector lens unit 15. An exit end face 16b of the light guide fiber 16 is situated close to the specimen so that the illumination light passing through the light guide fiber 16 and emerging from the exit end face 16b is radiated from an oblique direction toward the specimen 7 by the illumination lens unit 17. In the stereomicroscope of FIG. 2, the transmitted image of the specimen 7 need not be observed, and thus an opaque member is used for a stage 13.
Recently, in the field of biology, fluorescent pigment referred to as GFP (green fluorescent protein) has been come into prominent use. This pigment, in contrast with conventional fluorescent pigment, has the features that brightness is good and little bleaching is caused, and in addition, has the advantage of inflicting little damage on a living body. Consequently, in the fields of genetics and embryology, comprehensive research on the pigment is being done in such a way that the GFP pigment is applied, for example, to rats and fruit flies.
Of various specimens stained with the GFP pigment, specimens of rats and fruit flies such as in the foregoing are too large for optical microscopes and thus it is difficult to observe them with the optical microscopes. Hence, the needs of microscopes in which fluorescence observations can be made not only in a microregion but also in a macroregion are increased, and special attention has been devoted to stereomicroscopes for fluorescence observation in which a stereomicroscope having a long working distance and allowing a stereoscopic observation to be made is combined with a reflecting fluorescence device. Naturally, in ordinary fluorescence microscopes as well as in stereomicroscopes, applications of the GFP to fluorescence observations are becoming popular.
With a background of such needs, stereomicroscopes for making fluorescence observations, in addition to reflection observations and transmission observations, have recently been put to practical use. An example of a stereomicroscope which allows the fluorescence observation to be made is shown in FIG. 3. In this figure, like numerals are used for like members with respect to FIG. 1, and a detailed description of these members is omitted.
A conventional stereomicroscope in which the fluorescence observation can be made, as shown in FIG. 3, is provided with a transmitting illumination device C and a reflecting fluorescence illumination device D for fluorescence observation. The optical system of the transmitting illumination device C has members in common with that of the transmitting illumination device A, and includes the light source 9, a collector lens 18, a filter 19, a diffusion member 20, the deflection mirror 12, and the Fresnel lens 11. Illumination light emitted from the light source 9, after being rendered nearly parallel by the collector lens 18, passes through the filter 19, is diffused by the diffusion member 20, and is reflected by the deflection mirror 12 toward the specimen 7. The illumination light reflected by the deflection mirror 12 is changed to a light beam such that the specimen 7 is almost uniformly illuminated by the Fresnel lens 11. Where a color temperature is adjusted, the filter 19 is introduced into the optical path when necessary. On the other hand, the optical system of the reflecting fluorescence illumination device D includes a light source 21, an illumination lens 22, an excitation filter 23, a dichroic mirror 24L, and an absorption filter 25L.
Excited light emitted from the light source 21 which is a mercury lamp is introduced into the excitation filter 23 by the illumination lens 22. The excitation filter 23 selectively transmits only excited light with wavelengths required to excite the specimen 7, of the light from the light source 21. The excited light transmitted through the excitation filter 23 is reflected by the dichroic mirror 24L toward the zoom lens 2L and irradiates the specimen 7 through the zoom lens 2L and the objective lens 1.
The specimen 7 is such that because of the irradiation of the excited light, fluorescent light is emitted from parts stained by the fluorescent pigment. The fluorescent light originating from the specimen 7 is collected by the objective lens 1 and is introduced into the observation optical path R for the right eye and the observation optical path L for the left eye. The fluorescent light introduced into the observation optical path L for the left eye passes through the zoom lens 2L, is transmitted through the dichroic mirror 24L, and is selectively absorbed by the spectral characteristic of the absorption filter 25L so that only fluorescent light with particular wavelengths is transmitted therethrough. The fluorescent light with particular wavelengths is imaged by the imaging lens 3L and is observed as a fluorescent image through the eyepiece 4L. The fluorescent light introduced into the observation optical path R for the right eye, on the other hand, passes through the zoom lens 2R and a dichroic mirror 24R and reaches an absorption filter 25R. The fluorescent light transmitted through the absorption filter 25R with the same behavior as the absorption filter 25L is imaged by the imaging lens 3R and is observed as a fluorescent image through the eyepiece 4R.
The fluorescent light, in contrast with light in an ordinary reflection or transmission observation, is very low in intensity, and thus in various microscopes used for the fluorescence observation, irrespective of the stereomicroscopes in which the fluorescence observation can be made, it is very important to enable the fluorescent image of the specimen to be observed with a good contrast.
Thus, Japanese Patent Preliminary Publication No. Hei 9-292572 is such that a reflecting surface is provided below a specimen and thereby excited light transmitted through the specimen is reflected by the reflecting surface and illuminates again the specimen. By such an arrangement, the intensity of the excited light is doubled and a bright fluorescent image can be observed. On the other hand, Japanese Patent Preliminary Publication No. Hei 9-292570 proposes the means that in the transmitting fluorescence microscope, a long-wavelength cutoff filter and a short-wavelength cutoff filter are interposed between a specimen and a condenser lens and between the specimen and an objective lens, respectively. The long-wavelength cutoff filter prevents auto-fluorescent light produced by an illumination optical system from entering the objective lens, while the short-wavelength cutoff filter prevents excited light from entering the objective lens. Consequently, the amount of auto-fluorescent light superposed on the fluorescent image of the specimen can be diminished so that the fluorescent image with a good contrast is obtained.
Since each of conventional stereomicroscopes in which the fluorescence observation can be made, such as those mentioned above, is designed so that the fluorescence observation and the transmission observation are carried out, it is very useful and effective for the observation of a transparent specimen. The conventional stereomicroscope in which the fluorescence observation can be made, however, is nothing but a stereomicroscope in which the reflecting fluorescence illumination device D is merely added to the conventional stereomicroscope for transmission observation such as that shown in FIG. 1. Hence, a good fluorescence observation is not always carried out.
In other words, the conventional stereomicroscope in which the fluorescence observation can be made is such that the reflecting fluorescence illumination device is added to the stereomicroscope for transmission observation, and thus, for the glass stage, the lens member, and the diffusion member, optical members used in the conventional stereomicroscope for transmission observation have been used as they are. Specifically, from the viewpoint that manufacturing cost is kept to a minimum, an inexpensive green glass plate is used for the glass stage and resin materials are used for the lens member and the diffusion member of the transmitting illumination device.
However, where these optical members are illuminated with excited light transmitted through the specimen or radiated around the specimen, they have the drawback of considerably producing auto-fluorescent light, compared with glass producing little auto-fluorescent light which is used in an ordinary fluorescence microscope.
In particular, for the fluorescence observation in which the GFP pigment is used, the wavelength of the auto-fluorescent light produced by each of the optical members is not very different from that produced by the GFP pigment. Hence, it is difficult to separate the fluorescent light produced by the GFP pigment from the auto-fluorescent light by the dichroic mirror and the absorption filter which are arranged in each observation optical path. Consequently, the auto-fluorescent light is superposed as a background on the fluorescent light of the specimen, and the problem arises that the contrast of the specimen is considerably deteriorated.
In the stereomicroscope, a numerical aperture in a combination of a 1.times. objective lens with the zoom lenses is as low as about 0.1 at a magnification of 10.times., and a fluorescent image to be observed is very dark compared with the case where the numerical aperture in the fluorescence microscope is about 0.4 at almost the same magnification. In this way, the S/N ratio is highly limited, and a fluorescent image with a good contrast cannot be obtained.
The stereomicroscope which allows the fluorescence observation to be made has the problem that the observation images for the eyes are different in background brightness, due to the auto-fluorescent light from the stage glass and the optical member of the transmitting illumination system, caused by the excited light. This problem will be explained below with reference to FIG. 3.
In FIG. 3, excited light originating from the light source 21 is conducted to the zoom lens 2L of the observation optical path L for the left eye, of the two zoom lenses 2L and 2R. The excited light irradiates the specimen 7, and after being transmitted through the specimen 7, is also transmitted through the glass stage 8 to enter the transmitting illumination device C. In the transmitting illumination device C, auto-fluorescent light is emitted, due to the excited light, from the glass stage 8, the Fresnel lens 11, and the diffusion member 20. This auto-fluorescent light enters the objective lens 1 through the specimen 7. In this case, the zoom lens 2L of the observation optical path L for the left eye, in contrast with the zoom lens 2R of the observation optical path R for the right eye, is such that rays of the auto-fluorescent light are easy to enter.
Specifically, as shown in FIG. 3, the two observation axes 5L and 5R, after crossing at the specimen 7, is separated in the transmitting illumination device C. Here, since the excited light incident on the transmitting illumination device C travels along the observation optical axis 5L, the excited light with which the optical members such as the Fresnel lens 11 and the diffusion member 20 are irradiated is situated close to the observation optical axis 5L. Thus, the auto-fluorescent light is produced in the vicinity of the observation optical axis 5L, and most of the auto-fluorescent light is to be incident on the observation optical path L for the left eye with the observation optical axis 5L. As a result, the background of the observation optical path L for the left eye becomes bright. On the other hand, the observation optical axis 5R in the transmitting illumination device C is separated from the excited light, so that most of the auto-fluorescent light is not incident on the observation optical path R for the right eye. Consequently, the fluorescent image for the right eye is not affected by the auto-fluorescent light produced in the transmitting illumination device C, and the background darkens.
As mentioned above, since the conventional stereomicroscope for fluorescence observation is designed so that reflecting fluorescence illumination is provided from one of a pair of observation optical paths, the background of an observation image of the observation optical path where the reflecting fluorescence illumination is provided becomes bright, while the background of the other observation image becomes dark. It is, therefore, difficult for a viewer to integrally observe the left- and right-hand images, not to speak of a stereoscopic observation which is one of the features of the stereomicroscope. Thus, the problem arises that even in the case of a stereoscopic, simultaneous observation of the transmitted image due to the transmission illumination and the fluorescent image, it is very difficult to hold the entire image of the specimen.
Even with the use of a means for illuminating the specimen 8 with the excited light through the light guide, not through the zoom lens 2R or 2L, the same problem is raised.
In the case of a special frame for reflection illumination in which no optical system is provided below a specimen, it is common practice to use an opaque stage insert plate, in which, typically, one surface is white and the other is black. Where the stage insert plate is used for the reflecting fluorescence observation, a contrast is considerably deteriorated when the white side of the stage insert plate is placed below the specimen surface. Even when the black side is placed below the specimen, the auto-fluorescent light is emitted from the black stage insert plate, depending on the wavelength region of the excited light and kinds of resin material and paint of the stage insert plate, with a resulting deterioration in contrast.
In the discussion so far, reference has been made to the problems of auto-fluorescence in the stereomicroscope for fluorescence observation. However, even in the reflecting fluorescence observation of the conventional fluorescence microscope, the problems of the auto-fluorescence may be caused, as in the above description, by the optical members of the transmitting illumination device.
The conventional fluorescence microscope is designed so that, in the fluorescence observation, a light-blocking plate for intercepting excited light passing through a specimen is placed in a stage to thereby prevent the excited light from entering a condenser lens. With such an arrangement, however, the light-blocking plate must be removed from the illumination optical path each time the transmitting observation is carried out separately from the reflecting fluorescence observation, and thus working efficiency will be impaired. Moreover, even in a simultaneous observation for reflection fluorescence and transmission differential interference, the light-blocking plate must be removed from the illumination optical path. Consequently, the excited light caused by the reflection fluorescence passes through the specimen and enters the condenser lens, and the background of an observation image is rendered bright by auto-fluorescent light from the condenser lens, with a resulting deterioration in contrast. In order to suppress the emission of the auto-fluorescent light from the condenser lens, if the condenser lens is constructed of glass material in which little auto-fluorescent light is produced, the design of a condenser lens with high numerical aperture becomes difficult, and the degradation of aberration performance or an increase of the number of lenses will be caused, resulting in higher cost.
With the arrangement disclosed in Hei 9-292572, the brightness of the fluorescent image is increased nearly twice, but the intensity of auto-fluorescent light emitted from an objective lens is also increased nearly twice because the excited light reenters the objective lens. This constitutes an obstacle to the improvement of the S/N ratio and therefore the improvement of the contrast.
In Hei 9-292570, a technique of improving the contrast is disclosed. With this technique, however, auto-fluorescent light emitted from the illumination optical system and the objective lens can be cut off, but a sufficient space for placing the short-wavelength cutoff filter is not provided when the objective lens is short in working distance. Thus, in the case where the short-wavelength cutoff filter is constructed of colored glass, if the thickness of the filter is made small, the excited light cannot be completely cut off, while if it is larger, the aberration performance of the objective lens will be degraded. Furthermore, the contrast will be deteriorated by auto-fluorescent light from the colored glass filter. With the above technique, it is difficult to obtain a sufficient contrast. In addition, the use of the technique, which refers to transmitting fluorescence illumination, is not common practice.